Systems Modeling of Ca Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca²⁺ signaling in PC12 cells. We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca²⁺ signaling of bradykinin, a typical G_q- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca²⁺ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca²⁺ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn²⁺ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca²⁺ signaling.     

Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitochondrial Ca2+ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called 'calciosome'. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of approximately 10(-7) mol/l to approximately 10(-6) mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-insensitive Ca2+ pools Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a Ca2+/H+ exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

Store-Operated Ca2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

Aims

Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and Results

We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca²⁺ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions

These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca²⁺ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Molecular Biophysics of Orai Store-Operated Ca2+ Channels

Upon endoplasmic reticulum Ca²⁺ store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca²⁺-selective, Ca²⁺ release-activated Ca²⁺ (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

Modeling stochastic Ca2+ release from a cluster of IP3-sensitive receptors

We focused our attention on Ca(2+) release from the endoplasmic reticulum through a cluster of inositol(1,4,5)-trisphosphate (IP(3)) receptor channels. The random opening and closing of these receptors introduce stochastic effects that have been observed experimentally. Here, we present a stochastic version of Othmer-Tang model (OTM) for IP(3) receptor clusters. We address the average behavior of the channels in response to IP(3) stimuli. In our stochastic simulation we found that the fraction of open channels versus [IP(3)] follows a Hill curve, whose associate Hill coefficient increases when intracellular Ca(2+) level increase. This finding suggests that feedback from cytosolic Ca(2+) plays a key role in the channel response to IP(3). We also study several aspects of the stochastic properties of Ca(2+) release and we compare with experimental observations.     

Ca2+ uptake and IP3-induced Ca2+ release in permeabilized human lymphocytes

The 45Ca2+ uptake and 45Ca2+ release in saponin-permeabilized human lymphocytes were studied. An ATP-dependent Ca2+ uptake into a nonmitochondrial, intracellular Ca2+ store is observed which is approx. 2 orders of magnitude greater than the ATP-independent Ca2+ uptake. The Ca2+ uptake is inhibited by vanadate, but it is insensitive to oligomycin and ruthenium red. IP3 induces dose-dependent 45Ca2+ release. For half-maximum Ca2+ release 0.25-0.5 microM IP3 is required. The results of our studies suggest that 45Ca2+ is predominantly stored within the endoplasmic reticulum of the lymphocytes.     

Mobilization of mitochondrial Ca2+ by hydroperoxy-eicosatetraenoic acid

Hydroperoxy-eicosatetraenoic acids (HPETEs) and less effectively, also hydroxy-eicosatetraenoic acids (HETEs) stimulated Ca2+ release from rat liver mitochondria. Ca2+ release is accompanied by intramitochondrial pyridine nucleotide oxidation and hydrolysis. Both Ca2+ release and pyridine nucleotide oxidation are impeded when the flow of electrons between pyridine nucleotides and HPETE is impaired. Measurements of the mitochondrial membrane potential indicate that HPETE-stimulated Ca2+ release is not due to uncoupling of mitochondria. It is suggested that HPETEs and HETEs may act as mobilizers of mitochondrial Ca2+ during signal transduction related to proliferation and tumor promotion.     

Store-Operated Ca2+ Entry Is Remodelled and Controls In Vitro Angiogenesis in Endothelial Progenitor Cells Isolated from Tumoral Patients

Background

Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca²⁺ entry (SOCE), which is activated by a depletion of the intracellular Ca²⁺ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca²⁺-sensor, Stim1, and the plasmalemmal Ca²⁺ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca²⁺ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.

Methodology/Principal Findings

The present study employed Ca²⁺ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La³⁺ and Gd³⁺. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca²⁺ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca²⁺ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.

Conclusions

SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.     

IP3R, store-operated Ca2+ entry and neuronal Ca2+ homoeostasis in Drosophila

The IP3R (inositol 1,4,5-trisphosphate receptor) releases Ca2+ from the ER (endoplasmic reticulum) store upon binding to its ligand InsP3, which is thought to be generated by activation of certain membrane-bound G-protein-coupled receptors in Drosophila. Depletion of Ca2+ in the ER store also activates SOCE (store-operated Ca2+ entry) from the extracellular milieu across the plasma membrane, leading to a second rise in cytosolic Ca2+, which is then pumped back into the ER. The role of the IP3R and SOCE in mediating Ca2+ homoeostasis in neurons, their requirement in neuronal function and effect on neuronal physiology and as a consequence behaviour, are reviewed in the present article.     

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP₃‐generating agonists by producing Ca²⁺ waves. Here, the mechanism of wave progression has been investigated in voltage‐clamped single smooth muscle cells using localized photolysis of caged IP₃ and the caged Ca²⁺ buffer diazo‐2. Waves, evoked by the IP₃‐generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) over a single though substantial length (～30 µm) of the cell. During regenerative propagation, the wave‐front was about 1/3 the length (～9 µm) of the initiation site. The wave‐front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP₃ may explain the amplitude changes. Ca²⁺ was required for IP₃‐mediated wave progression to occur. Increasing the Ca²⁺ buffer capacity in a small (2 µm) region immediately in front of a CCh‐evoked Ca²⁺ wave halted progression at the site. However, the wave front does not progress by Ca²⁺‐dependent positive feedback alone. In support, colliding [Ca²⁺]_c increases from locally released IP₃ did not annihilate but approximately doubled in amplitude. This result suggests that local IP₃‐evoked [Ca²⁺]_c increases diffused passively. Failure of local increases in IP₃ to evoke waves appears to arise from the restricted nature of the IP₃ increase. When IP₃ was elevated throughout the cell, a localized increase in Ca²⁺ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP₃ and Ca²⁺ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley‐Liss, Inc.     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

IP3 receptors and their regulation by calmodulin and cytosolic Ca2+

Inositol 1,4,5-trisphosphate (IP(3)) receptors are tetrameric intracellular Ca(2+) channels, the opening of which is regulated by both IP(3) and Ca(2+). We suggest that all IP(3) receptors are biphasically regulated by cytosolic Ca(2+), which binds to two distinct sites. IP(3) promotes channel opening by controlling whether Ca(2+) binds to the stimulatory or inhibitory sites. The stimulatory site is probably an integral part of the receptor lying just upstream of the pore region. Inhibition of IP(3) receptors by Ca(2+) probably requires an accessory protein, which has not yet been unequivocally identified, but calmodulin is a prime candidate. We speculate that one lobe of calmodulin tethers it to the IP(3) receptor, while the other lobe can bind Ca(2+) and then interact with a second site on the receptor to cause inhibition.